Application of antigen gene and polypeptide thereof in preventing goose parvovirus disease

Abstract

The invention discloses an antigen gene and application of polypeptide thereof in preventing goose parvovirus disease. The antigen gene and the polypeptide provided by the invention can be used as a neutralizing epitope protein vaccine and a neutralizing epitope gene vaccine; the neutralizing epitope protein vaccine is a protein composed of 305 amino acid residues; a gene for coding the antigen polypeptide contains 918 nucleotides. The antigen gene and the polypeptide, namely the neutralizing epitope protein vaccine and the neutralizing epitope gene vaccine, provided by the invention have the advantages of strong antigenicity, capability of inducing an organism to generate a higher neutralizing antibody, good prevention effect, no pathogen transmission and the like, and a new situation is opened up in the GP prevention history.

Description

Technical Field

[0001] The invention relates to the application of an antigen gene and a polypeptide thereof as a vaccine in preventing goose parvovirus disease. Background Art

[0002] Gosling plague (GP) is a highly contagious, septicemic infectious disease caused by goose parvovirus (GPV). It is characterized by exudative enteritis, rapid transmission, strong pathogenicity and high mortality. It mainly attacks goslings and Muscovy ducks under 20 days old.

[0003] At present, vaccination is the main means of preventing GP. Attenuated vaccines have high immunogenicity, but are prone to virulence reversion and toxin shedding, and are dangerous to susceptible animals. Inactivated vaccines are highly safe, but have slow onset, a long window period, and a short immune protection period. The genome of GPV encodes three different structural proteins, namely VP1, VP2, and VP3, which are the key to determining viral tropism, host domain, and pathogenicity. Among them, VP2 protein is the main immune function area, which can stimulate the body to produce neutralizing antibodies. Therefore, it is very important to use genetic engineering technology to obtain a dense and protective neutralizing epitope vaccine, which can fundamentally eliminate the spread of pathogens. The present invention is to connect the protective antigenic epitopes of the VP2 protein identified in this experiment in series to prepare neutralizing epitope protein vaccines and gene vaccines, laying the foundation for the effective prevention of GP. Summary of the invention

[0004] The purpose of the present invention is to provide an antigen gene and its polypeptide, and the use of the antigen gene and polypeptide as a neutralizing epitope protein vaccine and a neutralizing epitope gene vaccine in preventing goose parvovirus disease. After the goose flock is immunized with the neutralizing epitope protein vaccine, the antibody level of the neutralizing epitope protein vaccine reaches a peak at 14 days, and the antibody level is still maintained at a high level at 35 days; while the antibody level of the neutralizing epitope gene vaccine reaches a peak at 21 days after immunization, and the antibody titer is always maintained at a high level. At the CD4 and CD8 transcription levels, both the neutralizing epitope protein vaccine group and the neutralizing epitope gene vaccine group are higher than the VP2 full-length protein group and the commercial vaccine group, indicating that the neutralizing epitope vaccine can stimulate the body to produce a stronger immune response. The results of the attack experiment show that the neutralizing epitope protein vaccine and the gene vaccine can effectively stimulate the body to produce an immune response and resist the invasion of viruses.

[0005] The genomic nucleotide sequence and protein sequence of the goose parvovirus disease neutralizing epitope protein vaccine and neutralizing epitope gene vaccine are within the protection scope of the present invention.

[0006] The antigen gene and polypeptide constructed by the present invention, namely the neutralizing epitope protein vaccine and neutralizing epitope gene vaccine for goose parvovirus disease, are obtained by using the neutralizing antibodies VHH18, VHH93, VHH96 and VHH95 stored in the laboratory to locate the neutralizing antigen epitope of VP2 gene (GenBank sequence number is No. KC996729.1), and then connecting them in series to express the obtained antigen gene and protein;

[0007] The neutralizing epitope vaccine is as follows (a) or (b): (a) a protein consisting of amino acid residues 1 to 305 from the N-terminus of sequence 2 in the sequence list; (b) a protein derived from (a) with the same activity after one or more amino acid residues are replaced and / or deleted and / or added;

[0008] The gene encoding the neutralizing epitope vaccine also falls within the protection scope of the present invention.

[0009] In the gene, the DNA molecule encoding the neutralizing epitope vaccine is as follows (1) or (2) or (3): (1) a DNA molecule shown in nucleotides 1 to 918 from the 5' end of sequence 1 in the sequence list; (2) a DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and encodes a protein with the same activity; (3) a DNA molecule that has at least 90% homology with the DNA sequence defined in (1) and encodes a protein with the same activity.

[0010] The above stringent conditions may be hybridization at 65°C in a solution of 6×SSC, 0.5% SDS, followed by washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0011] The expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the above-mentioned genes all fall within the protection scope of the present invention.

[0012] The related preparations of the goose parvovirus disease neutralizing epitope protein vaccine and neutralizing epitope gene vaccine are within the protection scope of the present invention.

[0013] The application of the antigen gene and polypeptide in preventing goose parvovirus disease is within the protection scope of the present invention.

[0014] The goose parvovirus disease neutralizing epitope vaccine constructed by the present invention can be regarded as a subunit vaccine and gene vaccine after genetic engineering modification, and its corresponding preparations, such as freeze-dried powder injection preparations, liquid vaccine preparations, etc., all fall within the scope of protection of this patent.

[0015] The present invention provides an antigen gene and its polypeptide, namely, a neutralizing epitope protein vaccine and a neutralizing epitope gene vaccine, which can stimulate the body to produce higher levels of neutralizing antibodies, achieve better protection effects, and improve the efficiency of attack and protection. It also has the advantages of strong antigenicity, controllable industrial production quality, high yield, and avoidance of pathogen transmission. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a neutralization epitope mapping map.

[0017] Figure 2 This is a DNA gel image of the construction of the neutralizing epitope vaccine. M1 is a molecular weight marker, and lane 1 is the gene fragment of the neutralizing epitope protein vaccine. M2 is a molecular weight marker, and lane 2 is the gene fragment of the neutralizing epitope gene vaccine.

[0018] Figure 3 The SDS-PAGE diagram of the neutralizing epitope protein vaccine. M is the molecular weight marker, and lanes 1 and 2 are the protein molecules of the neutralizing epitope protein vaccine.

[0019] Figure 4 This is a Western Blot image of the neutralizing epitope protein vaccine. Lane 1 is the protein of the neutralizing epitope vaccine, and lane 2 is the negative control.

[0020] Figure 5 This is the immunofluorescence image of the neutralizing epitope gene vaccine.

[0021] Figure 6 The antibody titers of experimental goslings immunized with neutralizing epitope protein vaccine and neutralizing epitope gene vaccine.

[0022] Figure 7 This is the mRNA transcription level of CD8 after experimental goslings were immunized with neutralizing epitope protein vaccine and neutralizing epitope gene vaccine.

[0023] Figure 8 This is the mRNA transcription level of CD4 after experimental goslings were immunized with neutralizing epitope protein vaccine and neutralizing epitope gene vaccine. Specific implementation plan

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0025] Example 1: Localization of Antigen Neutralizing Epitopes

[0026] The expression level of truncated proteins (V1-V11) in the periplasm of cells was detected by FCM. We transformed E. coli DH5α cells with pAPEx-VP2 plasmid and added positive colonies to liquid culture medium for culture. When the OD value 600 was 0.4-0.6, IPTG was added for induction. After culturing at 37°C for 4 hours, spheroplasts were prepared according to standard procedures and incubated with FITC-anti-Flag IgG (1:2000) and 1% BSA for 1 hour, and detected by FCM. A negative control was also set up. According to the expression level of V1-V11 protein in the periplasmic cavity of the bacterial inner membrane, different volumes of bacterial liquid were collected, and spheroplasts were prepared. They were incubated with 2μg VHH, anti-his antibody (1:500) and FITC-labeled goat anti-mouse antibody (1:2000) respectively, and then detected by FCM.

[0027] The results are as follows Figure 1 As shown, the neutralizing epitope of VHH18 is V1 and V2; the neutralizing epitope of VHH93 is V6; the neutralizing epitope of VHH96 is V9 and V11; and the neutralizing epitope of VHH95 is V3.

[0028] Example 2: Construction and identification of neutralizing epitope protein vaccines and gene vaccines

[0029] Primers were designed based on the six segments of neutralization epitope vaccine genes selected according to the positioning of the neutralization antigen epitope. Using the VP2 gene as a template, the six segments of the neutralization epitope vaccine genes were connected together using the overlap PCR method. The neutralization epitope vaccine gene fragments were recovered using a gel recovery kit, and the neutralization epitope vaccine gene fragments were connected to the pET27b vector and the pcDNA3.4 vector to construct a recombinant plasmid.

[0030] The results are as follows Figure 2 As shown, the gene size of the neutralizing epitope protein vaccine and the gene vaccine was approximately 918 bp, and the recombinant plasmid was successfully constructed.

[0031] Example 3: Expression and purification of neutralizing epitope protein vaccine

[0032] The obtained recombinant plasmid was introduced into Escherichia coli Rosetta to obtain recombinant bacteria. The obtained recombinant bacteria were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 120 r / min shaking until OD 600nm=0.35; add IPTG to a final concentration of 0.25mmol / L, and shake and culture at 37℃ and 120r / min for 4h. Centrifuge the bacterial solution at 4℃ and 4000r / min for 30min, and collect the bacterial precipitate. Resuspend the bacterial precipitate with PBS solution, add lysozyme solution (purchased from Amresco) and make the lysozyme concentration 1mg / mL, place at 4℃ for 1h, then perform ultrasonic disruption (25 watts of power, 3min), centrifuge at 4℃ and 10000g for 30min, and collect the precipitate. Wash the precipitate with washing solution 3 times, dissolve the precipitate with denaturing solution, and place it at 4℃ overnight. Add the denatured protein dropwise to the 10-fold system refolding solution and place it at 4℃ overnight. Dialyze the refolded protein into PBS solution, filter it, and store it in a -70 refrigerator.

[0033] The results are as follows Figure 3 As shown, the neutralizing epitope protein vaccine is expressed in the form of inclusion bodies, and purified protein can be obtained by denaturation and renaturation. The purity is over 90%, and its size is about 32 kDa.

[0034] Example 4: Identification of Neutralizing Epitope Protein Vaccines and Gene Vaccines

[0035] The purified VP2M protein samples were subjected to SDS-PAGE detection using 15% acrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in PBS at 37°C for 2 hours. Then, they were incubated with mouse anti-VP2 polyclonal antibody (1:2000), mouse anti-IL2 polyclonal antibody (1:500) and mouse anti-Flic polyclonal antibody (1:1000) at 37°C for 1 hour, and then incubated with HRP-labeled goat anti-mouse IgG (1:7500) at 37°C for 1 hour. Finally, the membranes were exposed using ECL luminescent detection reagents.

[0036] The obtained recombinant plasmid pcDNA3.4-VP2M was transfected into BHK-21 cells using Lipofectamine 3000 (Invitrogen, USA). The cells were incubated at 37°C and 5% carbon dioxide for 48 hours, the medium was discarded, washed twice with PBST, and 100 μL of cold propanol was added at -20°C for 1 hour; after washing with PBST, mouse anti-VP2 polyclonal antibody (1:2000) was added dropwise and incubated for 0.5 hours; after washing three times with PBST, 1:4000 goat anti-mouse secondary antibody (FITC labeled) was added dropwise, and the cells were observed under a fluorescence microscope after 30 minutes of reaction in the dark.

[0037] The results are as follows Figure 4 As shown in the results, mouse anti-VP2 polyclonal antibody can specifically recognize VP2M protein, and VP2M protein has good immunogenicity. Figure 5As shown in the figure, specific green fluorescence appeared 48 hours after pcDNA3.4-VP2M plasmid was transfected into BHK-21 cells, and the empty vector control group and negative control group had no specific green fluorescence. Therefore, the recombinant plasmid can express the target protein in BHK-21 cells and has good reactogenicity.

[0038] Example 5: Determination of antibody titer and antibody neutralizing activity after immunization of goslings with neutralizing epitope vaccines

[0039] 140 7-day-old commercial goslings were randomly divided into 7 groups, 20 in each group. Each gosling in the first group was subcutaneously injected with 4.34nmol of neutralizing epitope protein vaccine, each gosling in the second group was subcutaneously injected with 4.34noml of VP2 full-length protein vaccine, each gosling in the third group was subcutaneously injected with 100ug of neutralizing epitope gene vaccine, each gosling in the fourth group was subcutaneously injected with 100ug of full-length VP2 gene vaccine, each gosling in the fifth group was subcutaneously injected with 100μg of pcDNA3.4 gene vaccine, each gosling in the sixth group was subcutaneously injected with 100μL of normal saline, and each gosling in the seventh group was subcutaneously injected with 100μL of commercial vaccine. Every 7 days after immunization, venous blood was collected from each group of goslings to separate serum.

[0040] The antibody titer of each group of goslings was tested by ELISA. Figure 6 . The ELISA results showed that the antibody levels of the neutralizing epitope vaccine protein vaccine and gene vaccine groups after immunization first increased and then decreased. The antibody levels of the two vaccines always maintained high antibody titers throughout the entire immunization cycle. The neutralizing epitope protein vaccine took effect faster, and the neutralizing epitope gene vaccine could stimulate the body to produce antibodies for a long time, and the antibody titer was higher than that of other experimental groups.

[0041] Leukocytes were isolated from venous blood collected 3, 7, 10, 14, and 21 days after immunization and diluted to 5*10 6 A single cell suspension of 100 cells / ml was inoculated in a 48-well plate. After the cells were incubated for 24 hours, antigen protein was added for stimulation and culture for 24 hours, and the supernatant and cells were collected and PBS control was set up. Finally, the total RNA was extracted using the Trizol method, and the transcription levels of CD4 and CD8 were detected using the SYBR Green I fluorescent quantitative PCR method.

[0042] Results Figure 7 and Figure 8 After immunization with the neutralizing epitope vaccine protein vaccine and gene vaccine groups, the CD4 and CD8 transcription levels first increased and then decreased. The neutralizing epitope vaccine enhanced the body's immune response ability.

[0043] Example 6: Challenge test

[0044] At 14 days after immunization with the neutralizing epitope protein vaccine and 28 days after immunization with the epitope gene vaccine, the test geese were challenged with the virus and protected against the virus. The virulent strain H was selected and 100 ELD was injected subcutaneously into each goose. 50 Dosage for attack.

[0045] Ten days after the challenge, the number of gosling deaths in each group was counted. The results are shown in Table 1.

[0046]

Claims

1. An antigen gene and its polypeptide, which is a gene obtained by selecting 6 segments of neutralizing epitopes in the VP2 gene and connecting them in series, and a protein obtained by expressing them; The antigen polypeptide is as follows (a) or (b): (a) a protein consisting of amino acid residues 1-305 from the N-terminus of sequence 2 in the sequence list; (b) a protein derived from (a) with the same activity after one or more amino acid residues are replaced and / or deleted and / or added.

2. A gene encoding the antigenic polypeptide according to claim 1.

3. The gene according to claim 2, characterized in that: In the gene, the DNA molecule encoding the antigen polypeptide is the following (1) or (2) or (3): (1) the DNA molecule represented by nucleotides 1 to 918 from the 5' end of sequence 1 in the sequence list; (2) a DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and encodes a protein having the same activity; (3) A DNA molecule that has at least 90% homology with the DNA sequence defined in (1) and encodes a protein with the same activity.

4. The expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the gene described in claim 3 all fall within the protection scope of the present invention.

5. Use of the antigen polypeptide according to claim 1 and the gene of the antigen polypeptide according to claim 3 in the preparation of related products.

6. Use of the antigenic polypeptide according to claim 1 and the gene of the antigenic polypeptide according to claim 3 in preventing goose parvovirus disease.

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